Abstract
BY-kinases represent a highly conserved family of protein tyrosine kinases unique to bacteria without eukaryotic orthologs. BY-kinases are regulated by oligomerization-enabled transphosphorylation on a C-terminal tyrosine cluster through a process with sparse mechanistic detail. Using the catalytic domain (CD) of the archetypal BY-kinase, Escherichia coli Wzc, and enhanced-sampling molecular dynamics simulations, isothermal titration calorimetry and nuclear magnetic resonance measurements, we propose a mechanism for its activation and nucleotide exchange. We find that the monomeric Wzc CD preferentially populates states characterized by distortions at its oligomerization interfaces and by catalytic element conformations that allow high-affinity interactions with ADP but not with ATP·Mg(2+) We propose that oligomer formation stabilizes the intermonomer interfaces and results in catalytic element conformations suitable for optimally engaging ATP·Mg(2+), facilitating exchange with bound ADP. This sequence of events, oligomerization, i.e., substrate binding, before engaging ATP·Mg(2+), facilitates optimal autophosphorylation by preventing a futile cycle of ATP hydrolysis.